Low-pH adaptation and the acid tolerance response of Bifidobacterium longum biotype longum

Abstract

Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.

FIG. 1.

Representative 2D gels containing cytosolic extracts from mid-exponential-phase cells of B. longum biotype longum NCIMB 8809 (left) and B. longum biotype longum 8809dpH (right) grown in MRSC initially adjusted to pH 7. Spots identified by peptide mass fingerprinting are labeled, and the corresponding identifications are listed in Tables 2 and 3. Spots that showed variations between the WT and the mutant are underlined, whereas those that modified their quantities at pH 4.8 are in bold.

FIG. 2.

Schematic representation of the carbon catabolic pathway, the bifid shunt, and the BCAA metabolism. Enzymes whose production was modified after adaptation or exposure to acid pH in B. longum are marked with a gray circle.

FIG. 3.

Redox ratios (A), NADH-associated fluorescence (B), and FAD-associated fluorescence (C) of B. longum biotype longum NCIMB 8809 and B. longum biotype longum 8809dpH grown at neutral pH (solid bars) or acid pH (white bars). Vertical lines on the bars represent standard deviations.

FIG. 4.

(A) Intracellular pH of B. longum cells (grown in MRSC at pH 7.0) after suspension in buffer at pH 7.0 or pH 4.8. (B and C) Valine concentration (B) and NH₄⁺ concentration (C) measured in cell extracts of B. longum biotype longum NCIMB 8809 and B. longum biotype longum 8809dpH grown at neutral pH (black bars) or acid pH (grey bars).